Although spermatogonial stem cells are essential for reproduction, little is known about their regulation. Environmental and intrinsic factors are crucial for stem cell establishment and maintenance, but systematically identifying these factors is extremely challenging in mammalian systems. We use Drosophila spermatogenesis as a model system, since it parallels mammalian systems, yet we can precisely locate the stem cells, and manipulate their microenvironment genetically. Prior funding enabled us to discover the molecular mechanism controlling Drosophila spermatogonial stem cell self-renewal. Germline stem cells (GSCs) are anchored around a cluster of somatic support cells called the hub. The hub produces a ligand that activates Jak-Stat in adjacent germ cells, instructing them to remain as stem cells. Daughters displaced away from the hub differentiate. Building on preliminary data derived from these findings, in this renewal we address three fundamental questions. In Aim 1, we pursue our preliminary data suggesting that Jak-Stat signaling converts GSC precursors (primordial germ cells) into GSCs by characterizing germ cell behavior and Jak-Stat activity in the embryonic testis, then using genetics to determine the role of this pathway in GSC establishment. In Aim 2, we extend our studies of Jak-Stat signaling in adult stem cells. Although GSCs require direct activation of Stat, this was not tested for SSCs. In Aim 2a, we determine if Jak-Stat signaling is directly or indirectly required for GSC maintenance. In Aim 2b, we pursue our extensive preliminary data indicating that socs36E, a target and inhibitor of Jak-Stat signaling, maintains GSCs in the testis. We will complete the characterization of our socs36E alleles, then we will determine if socs36E is directly or indirectly required for GSC maintenance. Finally, since targets of Stat in GSCs (&possibly SSCs) may be intrinsic determinants of stem cell fate, while targets in hub cells may regulate hub cell function, we will identify these targets, and test them for roles in stem cell maintenance using genetic approaches in Aim 3. Together this work will provide fundamental insight into the molecular mechanisms that regulate stem cell establishment and maintenance, which is of great importance towards developing potential therapeutic uses of stem cells in regenerative medicine.